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1
Basiran, Mohd Nazir. "DNA insertion mutagenesis in higher plants." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35436.
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Themis, Michael. "Retrovirus insertional mutagenesis : experiences at the hprt locus." Thesis, Brunel University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307483.
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Bokhoven, Marieke Christina. "Measurement of insertional mutagenesis by retroviral and lentiviral vectors." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444113/.
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Retroviral vectors have been successfully used in the clinic for the correction of inherited immunodeficiencies. However, in 2 recent gene therapy trials for X-linked Severe Combined Immunodeficiency, 5 patients developed leukaemia. This was associated with vector integration near cellular proto-oncogenes, leading to their activation a process known as insertional mutagenesis. This thesis describes the development of a cell line assay that allows us to quantify insertional mutagenesis by retroviral and lentiviral vectors and to analyse the mechanisms by which this occurs. The interleukin-3 (IL-3) dependent cell line BCL15 is derived from the mouse bone marrow cell line BAF3 and over- expresses human Bcl2. The frequency at which IL-3 independent mutants are obtained following vector transduction is measured. Lentiviral and retroviral vectors transform BCL15 cells at similar integrant frequencies of 4.3 x 108 and 1.2 x 107, respectively. However, they cause insertional mutagenesis in this assay by different mechanisms. The human immunodeficiency virus-1 (HIV-1)- derived lentiviral vector HV transforms BCL15 cells by insertional activation of the growth hormone receptor (Ghr) gene. An HIV-Ghr fusion transcript was detected. It originates from the HIV-1 5'-LTR it then splices from the HIV-1 major splice donor to the splice acceptor of Ghr exon 2. The mutants express GHR and grow in response to bovine growth hormone in the foetal calf serum of the culture medium. Deletion of the HIV-1 enhancer/promoter in a self-inactivating vector prevents this mechanism of transformation. Retroviral vector transformation of the BCL15 cell line does not occur via Ghr gene activation. Retroviral vectors up- regulate expression of the cytokine IL-3, either by insertion into the IL-3 gene or by insertion into other genes, which may act as upstream activators of IL-3 expression. This assay is a general method to quantitate insertional mutagenesis. It may inform the design of safer vectors and can be used in initial safety testing of (pre-) clinical vectors.
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Gan, Shu Uin. "Retroviral insertional mutagenesis of human myeloid HL-60 cells." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244680.
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Gaiano, Nicholas R. (Nicholas Roger). "Insertional mutagenesis in zebrafish using a pseudotyped retroviral vector." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/43311.
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Kong, Jun. "Transposon-mediated insertional mutagenesis in gene discovery and cancer." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609377.
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Bronchain, Odile Jacqueline. "Insertional mutagenesis through gene trap approaches in Xenopus embryos." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620913.
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Sutherland, Helen F. "Gene targeting and insertional mutagenesis in embryonic stem cells." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/20231.
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Mutations are important in the study of gene function. Until recently mouse mutants available for study were spontaneous mutations and those derived from chemical or X-ray mutagenesis. Transgenic technology brought about the creation of more mouse mutants through insertional mutagenesis by retroviral agent or microinjected recombinant DNA. The insert may be used as a tag to molecularly clone and characterise the mutated gene. With the advent of embryonic stem (ES) cell technology the number of mutations available for study should grow - both specific and random mutations. ES cells are pluripotent stem cells, that may be manipulated genetically in culture, and yet retain the ability to contribute to normal development and the germ line of a host blastocyst. Thus the phenotypic effects of the introduced mutation may be studied in vivo. Random mutations may be introduced into the ES cell genome by insertional mutagenesis by retroviral agent or by promoter or enhancer trap vectors. Gene targeting by homologous recombination may be used to introduce specific mutations. Hox-2.1 is a mouse homeobox-containing gene, belonging to the family of Hox genes. It has been cloned and mapped to the Hox-2 cluster on chromosome 11. Hox genes are thought to play important roles in mouse development. They may act as regulatory genes, controlling the expression of structural genes at different positions in the developing embryo. In order to study the function of Hox-2.1 in development it would be useful to have a mouse mutant for Hox-2.1. It was the aim of this project to produce a mouse mutant for Hox2.1. The first step towards this aim is the targeting/knock-out of the gene in ES cells. During this project various Hox-2.1 targeting constructs have been designed and built. Replacement vectors, incorporating a promoterless neo or employing the positive-negative selection strategy, were designed to enrich for targeting events. These and two insertion vectors were transfected separately into ES cells. Over 200 selected clones have been screened for disruption of the Hox2.1 locus. No homologous recombinants were identified. One clone, known as a 'pick-up' clone, was identified. It is thought that the targeting vector has found homology with the target sequence, picked up DNA from the locus (in this case 3' to sequence within the construct) and integrated elsewhere in the genome. An integration site, which the promoterless Hox2.1-neo targeting vector has integrated into twice, despite non-homology, has been characterised. It is suggested that this locus may be a site of frequent integration. The sequence flanking the construct was amplified by inverse PCR, cloned and sequenced. The properties which may make it highly targetable are discussed. It was shown to have weak promoter function.
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Jiang, Xiaoyan. "Insertional mutagenesis by provirus in retrovirus-induced lymphoid murine tumors." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28790.
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Ahi-1 was initially identified as a common helper provirus integration site on mouse chromosome 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to Myb proto-oncogene. By using long-range restriction mapping, we have mapped the Ahi-1 locus approximately 35 kbp downstream of the Myb gene. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region may activate a novel gene, apparently involved in tumor formation. In addition, another provirus integration site which was identified in 14% of immature T-cell thymomas induced by Mo-MuLV infection of MMTV${{ rm D} over C}$-Myc transgenic mice has recently been mapped within the Ahi-1 region. We have used exon amplification to identify genes that may cooperate with v-Ab1 or c-Myc in B- and T-cell transformation. We have indeed identified a novel Ahi-1 gene which encodes two major RNA species of 2 kbp and 5 kbp. The Ahi-1 gene is expressed in several organs of the mouse and rat and is highly expressed in brain and testis. One cluster of the proviruses was found to integrate 3$ sp prime$ of noncoding region of the Ahi-1 in an inverse transcriptional orientation. The Ahi-1 is highly conserved in evolution and encodes a 297 amino acid protein. The predicted Ahi-1 protein contains a SH3 domain and four potential SH3-binding sites, which function to mediate specific protein-protein interactions during signaling. The human homologue of the Ahi-1 gene has been cloned and mapped to chromosome 6q 23-24, and located approximately 330 kbp downstream of the Myb gene. The Ahi-1 is a novel gene which may play important roles in the signal transduction and tumor development.Mis-2 and Mis-4 loci were identified as two new common provirus integration sites in Moloney MuLV-induced thymomas. Mis-2 has been mapped on mouse chromosome 10 and located 160 and 40 kbp downstream of Myb and Ahi-1 genes. The Mis-4 locus has been located on chromosome 15 at about 60 and 30 kbp downstream of Myc and Mlvi-4 genes. Myc RNA levels are elevated in tumors harboring a Mis-4 rearrangement compared with some of those with no Mis-4 rearrangement, indicating that provirus integration affected Myc expression by long-distance activation. Both loci may contain novel genes involved in tumor formation.
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Franz, Marie-Josee. "Analysis of factor-independent mutants induced by retroviral insertional mutagenesis." Thesis, Brunel University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262514.
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Turney, Catherine Louise. "Transposable elements of the mariner family in the tephritid fruit fly, Bactrocera tryoni." Thesis, The University of Sydney, 1998. https://hdl.handle.net/2123/27649.
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The research outlined in this thesis primarily focused on the isolation and
characterization of representatives of the mariner family of transposable
elements in the genome of the tephritid, Bactrocera tryoni (Queensland
fruit fly). A preliminary investigation was also made of the mobility
properties of constructs based on the mariner element Mos], following
their transient introduction into the embryonic soma of B. tryoni. This
involved the use of plasmid-based excision and transposition assays. These
studies were partly undertaken to obtain an initial idea of the feasibility of
developing a germline transformation system based on particular mariner
elements for use with B. tryoni, and possibly other related tephritids.
To investigate whether the B. tryoni genome contained endogenous
mariner element copies, an initial PCR analysis was carried out using
degenerate oligonucleotide primers that had previously been designed to
stretches of conserved residues within transposases encoded by particular
mariner elements. Using this approach, examples of at least five distinct
types of mariner elements were detected in the genome of B. tryoni
following the cloning and DNA sequencing of 20 unique amplified
fragments. Phylogenetic analyses using the conceptual amino acid
translations of these partial transposase gene regions indicated that the B.
tryom‘ sequences represented three subfamilies of mariner elements. Using
the same PCR-based approach, diverse mariner sequences were also
isolated from Bactrocera neohumeralis, a sibling species of B. tryoni, and
an additional tephritid, Bactrocera jarvisi.
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Chen, Weiping. "Generation of new colonial mutants in Neurospora crassa using insertional mutagenesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ57785.pdf.
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Mikkers, Henricus Martinus Maria. "Retroviral insertional mutagenesis and characterization of the frequently activated PIM kinases." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2003. http://dare.uva.nl/document/70366.
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Islam, Muhammad Sougatul. "Insertional mutagenesis to identify novel determinants of pathogenicity in Magnaporthe oryzae." Thesis, University of Exeter, 2012. http://hdl.handle.net/10871/10802.
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Rice blast disease is caused by the filamentous fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. It was the first plant pathogenic fungus to have its genome sequence published which opened up the opportunities to discern the principal genetic components that confer pathogenicity on the fungus. The availability of the genome sequence has also presented fresh challenges in terms of converting sequence data into meaningful biological information. Functional genomics studies involve the generation of genome-wide mutant collections and comprehensive screens with potential to identify novel pathogenicity determinants. In this study I utilized Agrobacterium tumefaciens mediated random insertional mutagenesis to study the infection mechanism of M. oryzae. A collection 10,200 M. oryzae T-DNA insertion mutants were generated as part of this study and pathogenicity was assayed by high-throughput disease screening. From the primary qualitative screening I obtained 200 mutants that were reduced or lacking in pathogenicity. Quantitative re-screening allowed selection of 71 T-DNA mutants, including 9 non-pathogenic and 63 reduced virulence mutants exhibiting at least a 50% reduction in disease symptoms. Finally, we selected 8 non-pathogenic mutants for detailed phenotypic and gene functional analysis. A novel approach was used to retrieve T-DNA tagged genes from mutants of interest. Next generation DNA sequencing (NGS) was used to retrieve T-DNA flanking sequences in a high-throughput manner. The efficiency of NGS to facilitate the high-throughput large scale insertional mutagenesis was therefore demonstrated. Out of 8 selected mutants, I identified three novel genes that putatively encode a transcription factor, a PH domain containing signalling protein and a MAP kinase. I also provided evidence that, MGG_05343 is a functional C6 zinc finger transcription factor involved in conidiogenesis. The PH domain containing protein MGG_12956 is involved in vegetative growth, condiogenesis and virulence. The novel kinase MGG_15325 is a S. cerevisiae IME2 homolog that belongs to the Ime2 class of non-classical MAP kinase subfamily. Intriguingly, M. oryzae IME2 seems to have an essential role in growth in planta because the mutant was able to penetrate and colonize plant tissue but failed to cause necrotic rice blast lesions. Identification of these novel genes will allow us greater insight into the processes required for condiogenesis, vegetative and invasive growth and a more integrated understanding of the post-penetration phases of plant tissue colonization. Interestingly, I identified two mutants tagged with T-DNA insertion in the autophagy genes ATG2 and ATG3, reaffirming the importance of infection-associated autophagy in plant infection by M. oryzae and we characterized the ATG3 gene. In addition, I generated a resource of 63 unidentified T-DNA mutants which can potentially lead to identification of more novel determinants of pathogenicity in rice blast disease.
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Chen, Weiping Carleton University Dissertation Biology. "Generation of new colonial mutants in Neurospora crassa using insertional mutagenesis." Ottawa, 2001.
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Paisley, Derek John. "Characterisation of two mouse mutant lines generated by transgene insertional mutagenesis." Thesis, University of Bath, 2000. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342073.
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Amsterdam, Adam (Adam Henry) 1967. "Use of a pseudotyped retoviral vector to accomplish insertional mutagenesis in zebrafish." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/50024.
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Trouvelot, Sophie. "Obtention et caractérisation, phénotypique et génotypique,de mutants de la souche Fusarium oxysporum Fo47,affectés dans leur aptitude antagoniste." Dijon, 2002. http://www.theses.fr/2002DIJOS048.
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Les souches non pathogènes de Fusarium oxysporum telles que Fo47 ont souvent la propriété de protéger la plante contre l'attaque de la souche qui lui est pathogène. Cette capacité repose sur des mécanismes de compétition pour les éléments nutritifs et les sites d'infection et sur l'induction de résistance chez la plante. Cependant, le déterminisme de ces modes d'action reste inconnu. Les objectifs de ces travaux ont été d'analyser les modes d'action de la souche Fo47 et d'identifier si possible des gènes impliqués dans ces processus. La démarche adoptée a consisté à obtenir des mutants affectés dans leur capacité à protéger, puis à les caractériser aux plans génotypique et phénotypique. N'ayant pas d'a priori sur les gènes dont les produits pourraient être impliqués dans l'activité protectrice, nous avons opté pour une stratégie de mutagenèse aléatoire : la mutagenèse insertionnelle par transposon. L'élément transposable Fot1 a été utilisé car la souche Fo47 ne possède aucune copie endogène de ce transposon. Par cette technique 12 mutants significativement affectés ont été obtenus et 2 ont été analysés sur les plans moléculaire, protéomique, saprophytique et dans l'interaction avec la planteBiological control of Fusarium wilts can be achieved by using nonpathogenic strains of F. Oxysporum selected for their antagonistic effect against pathogenic formae speciales. Three major mechanisms are generally reported, which may be present within the same biocontrol strain : competition for nutrients; competition for infection sites and root colonisation; induced systemic resistance. However, the determinism of these modes of action is unknown. The objectives of this work was to analyse the mechanisms implicated in the antagonistic activity of the strain Fo47 and to identify genes involved in this process. In this way, we decided to produce mutants impaired in their antagonistic activity, then to characterise them molecularly and phenotypically. Having no hypothesis about genes involved in this process, we opted for a so-called "black box" approach. We choose a transposon-mediated insertional mutagenesis strategy. In our work, we used the Fot1 element because Fo47 have no endogenous copy of this transposon. Thanks to this method, we obtained 12 mutants which were significantly affected in their biocontrol activity and 2 were characterized with different approaches : molecular, proteomic, saprophytic and in interaction with the host plant
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Berschback, John. "Application of a Unique Insertion Mutagenesis Method to Characterize Genes Responsible for Biofilm Formation by Acinetobacter baumannii." Miami University Honors Theses / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=muhonors1111088001.
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Villeneuve, Luc. "Insertional mutagenesis by provirus integration in Moloney murine leukemia virus-induced rat thymomas." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74060.
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Bellanger, Fabienne. "La mutagenèse par insertion d'ADN chez Chlamydomonas reinhardtii : une nouvelle voie pour l'élucidation de la biosynthèse." Compiègne, 1994. http://www.theses.fr/1994COMP686S.
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Une souche sans paroi de l'algue verte unicellulaire chlamydomonas reinhardtii auxotrophe pour l'arginine a été transformée par le plasmide parg 7. 8 par agitation en présence de billes de verre. Les cellules ayant intégré une copie fonctionnelle du plasmide ont été sélectionnées par leur capacité à croître sur un milieu dépourvu d'arginine, le plasmide portant le gène de l'argino-succinate lyase sauvage complémentant l'auxotrophie. Des souches déficientes pour la biosynthèse de l'amidon ont été sélectionnées parmi les transformants. Parmi les 16 souches présentant un phénotype mutant pour la synthèse d'amidon, 10 accumulent de très faibles quantités du polysaccharide (inferieure à 5% de la quantité mesurée chez la souche sauvage). Deux souches sont mutées pour la synthèse de l'amylose et 4 autres accumulent une amylopectine modifiée. Une analyse génétique de la souche BAF R1 a permis de mettre en évidence l'intégration d'un plasmide parg 7. 8 fonctionnel au locus ST-2, conduisant à la délétion du gène de structure de la GBSS (Granule Bound Starch Synthase). Ceci nous a permis de prouver que la synthèse de longs glucanes n'est pas un prérequis pour la synthèse de l'amylopectine. L'un des mutants pauvres en amidon ne présente pas d'activité ADP-GLC pyrophosphorylase détectable. Cette délétion complémente la mutation ST-1-1 précédemment caractérisée comme défectueuse pour la même activité enzymatique. Ceci suggère que la transformation a conduit à la mutation du deuxième gène de structure de l'ADP-GLC pyrophosphorylase. La présence d'un polysaccharide soluble fortement branché, de type phytoglycogène, a été détectée chez un autre des mutants pauvres en amidon. 50 souches ont également été sélectionnées suite à un maintien de l'amidon dans des conditions qui induisent sa dégradation chez le sauvage.
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Sveric, Krunoslav Michael. "Novel strategies for the identification of clock genes in Neurospora crassa with insertional mutagenesis." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-170499.
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Circadian clocks are endogenous cellular mechanisms that control daily rhythms of physiology and behaviour. The adjustment of the circadian clock to the 24 h period of a day is commonly accomplished by several environmental cues, e.g. temperature, light and nutrition. For one light input pathway the mechanism that synchronises or entrains Neurospora’s clock is supposed to be known.
Nevertheless, there are plenty more environmental cues that have an obvious impact on the circadian clock, e.g. temperature.
The environmental cue “temperature” was underrepresented in studies about Neurospora’s circadian clock, while several clock studies focused on stationary conditions rather than changing ones. As a result, the functionality and adaptation of circadian clocks were underestimated, and thus new clock components could be overlooked due to screening on constant darkness.
It was therefore important to develop a novel strategy in screening mutants that challenged the circadian clock of Neurospora crassa entirely on temperature alternations. A temperature cycle of low amplitude (22° C cold and 27° C warm) and of short period (8 h cold and 8 h warm) applied as Zeitgeber stimulus.
A mutant library was created with insertional mutagenesis via electroporation in order to transform a BASTA resistance gene into conidial nuclei. As a consequence, a novel method of rescuing the mutations was established, which
combined the process of mapping, partial cloning and PCR and was called Size Selected Fragment Plasmid Rescue or SSFPR in short.
During the screening of several hundred mutants among the novel protocol, a known clock gene, frequency, was identified and characterised. The identification of several mutants with altered clock phenotypes has on one hand confirmed the
general approach of this study and on the other proved that the greater sensitivity for the temperature screen can bee used to detect mutant phenotypes.
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Culp, Patricia Ann. "Random DNA integrations as an approach to insertional mutagenesis in the zebrafish (Brachydanio rerio)." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/33505.
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Indukuri, Vijaya Varma. "Transposon based mutagenesis and mapping of transposon insertion sites within the Ehrlichia chaffeensis genome using semi random two-step PCR." Thesis, Kansas State University, 2013. http://hdl.handle.net/2097/16329.
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Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Roman Reddy Ganta
Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism.
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Johansson, Fredrik. "Screening for Candidate Brain Tumor Genes : Identifying Genes that Cooperate with Platelet-Derived Growth Factor in Glioma Development and Progression." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7185.
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Tramontano, Andrea [Verfasser], George [Akademischer Betreuer] Coupland, and Martin [Akademischer Betreuer] Huelskamp. "Retrotransposon Tto1 : functional analysis and engineering for insertional mutagenesis / Andrea Tramontano. Gutachter: George Coupland ; Martin Huelskamp." Köln : Universitäts- und Stadtbibliothek Köln, 2011. http://d-nb.info/1038225078/34.
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Schulte, Cathleen Elizabeth. "Isolation and analysis of factor independent murine haematopoietic progenitor cells (FDCP-1) by retroviral insertional mutagenesis." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243333.
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Dasgupta, Maupali. "Screening for activators of NF-kB using Sleeping Beauty Transposons." Kent State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=kent1201807856.
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Fusil, Floriane. "Expansion in vivo des cellules génétiquement corrigées et sécurité oncongénique : application a la thérapie génique des hémoglobinopathies." Paris 7, 2008. http://www.theses.fr/2008PA077052.
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La thérapie génique par transplantation de cellules souches hématopoïétiques génétiquement modifiées peut constituer une thérapeutique idéale pour les patients atteints de maladies génétiques du système sanguin. Cette méthode a été validée entre autres par des résultats précliniques probants dans des modèles murins de β-thalassémie. Deux problèmes associés au transfert de gène dans les cellules hématopoïétiques doivent être résolus: 1) réduire le conditionnement toxique préalable à la greffe ; 2) évaluer et minimiser les risques de mutagenèse insertionnelle. En l'absence d'avantage sélectif spontané, aucun bénéfice thérapeutique ne peut être observé sans conditionnement myéloablatif. Deux systèmes, basés sur l'érythropoïétine et son récepteur, dont le but est d'amplifier in vivo les cellules érythroïdes génétiquement corrigées ont été évalués: une forme membranaire de l'érythropoïétine et un récepteur tronqué. Pour favoriser la prolifération des cellules érythroïdes modifiées, l'expression de l'érythropoïétine membranaire doit être réduite et se produire à un stade de différenciation érythroïde tardif. L'expression érythroïde du récepteur tronqué permet d'augmenter in fine la proportion de globules rouges corrigés. L'expansion est régulée de manière physiologique, par la concentration plasmatique d'Epo, elle-même corrélée au degré de correction de la pathologie. Ceci reflète un phénomène de contrôle physiologique de l'homéostasie hématopoïétique. La sécurité oncogénique d'un vecteur lentiviral (utilisé en clinique), produisant la chaîne p-globine thérapeutique, a été évaluée dans un modèle transgénique pour l'oncogène Spi1, prédisposé au développement d'érythroleucémiesGene therapy by transplantation of genetically modified hematopoietic stem cells would be ideal for patients with genetic blood diseases. The foremost advantage of infusing genetically modified autologous cells is to avoid the risk of graft-versus-host disease and the host immunosuppression necessary for preventing graft rejection. However, cytoreductive regimens are still required to achieve significant chimerism with small number of modified hematopoietic stem cells. One question is how to establish a stable chimerism with maximal repopulation by genetically modified cells, with minimal or without conditioning, with low number of corrected stem cells and with no risk of abnormal cell proliferation. We tested in vivo the cell expansion ability of two Systems based on erythropoietin and its receptor, especially adapted for gene therapy of hemoglobinopathies. A truncated EpoR, together with the p-globin transgene, induced a 100-fold expansion of erythroid cells in a mouse model of p-thalassemia. The second objective was to develop an acquired preleukemic mouse model in order to evaluate the oncogenic risk of a viral LentiGlobin vector for gene therapy of erythroid cell disorders. A toxicology study was performed in irradiated mice transplanted with bone marrow cells from donor transgenic mice overexpressing Spi-1 (commonly activated oncogene in Friend-mediated mouse erythroleukemias), with hightened susceptibility towards this hematological malignancy. The LentiGlobin vectors did not increase the leukemic risk
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Nannapaneni, Kishore. "Design of a bioinformatics system for insertional mutagenesis analysis and its application to the Sleeping Beauty transposon system." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1039.
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Cancer is one of the leading causes of death in the world. Approximately one fifth of deaths in the western industrial nations are caused by cancer. Every year several hundreds of thousands of new patients are diagnosed with cancer and several thousands die of cancer. Scientists have been conducting research from different angles for effective prevention, diagnosis and cure of Cancer.
Ever since the genetic basis of cancer has been demonstrated, a race has been ignited globally in the scientific community to identify potential oncogenes and tumor suppressor genes. The genetics of the tumors are complex in nature where combinations of loss of function mutations in tumor suppressor genes and gain of function mutations in oncogenes cause cancers. The identification of these genes is extremely important to devise effective therapies to treat cancer. Insertional mutagenesis systems such as sleeping beauty provide an elegant way to identify genes involved in cancers. More and more researchers are adopting the Sleeping Beauty system for their insertional mutagenesis experiments to identify potential cancer causing genes. Given next generation sequence technologies and the vast amount of data they generate requires novel bioinformatics techniques to process, analyze and meaningfully interpret the data. The goal of this project is to develop a publicly available system for researchers worldwide to analyze the sequence data resulting from insertional mutagenesis experiments.
This system will identify and annotate all the insertion sites resulting from the sequencing of the experiment. It will also identify the Common Insertion sites (CIS) and genes with Common Insertion Sites (gCIS). The Common Insertion Sites being the regions in the genome that are targeted more often than by chance. The whole system is accessible as a web application for use by researchers worldwide performing insertional mutagenesis experiments.
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BINDER, HEIDE-MARIE. "SLEEPING BEAUTY FINDS PARTNERS OF ONCOGENIC MYC - A TRANSPOSON-BASED INSERTIONAL MUTAGENESIS SCREEN IN THE EMU-MYC MOUSE." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234150.
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Myc triggers a transcriptional program inducing hyper-replication and proliferation but also tumor suppressive mechanisms like apoptosis. Therefore, Myc dependent tumors display high selective pressure to accumulate secondary mutations blocking these tumor-suppressive pathways. In the Eµ-myc mouse model, Myc is constitutively expressed in the B-cell linage under the control of the immunoglobulin heavy chain enhancer. The most prominent failsafe program known to be disrupted in Eµ-myc lymphomas is the p53-ARF pathway. In order to find novel cooperating partners of Myc leading to transformation we applied a conditional Sleeping Beauty transposon-based mutagenesis screen in vivo. By adoptive transplantation of Eµ-myc hematopoietic progenitors we generated 312 experimental animals, prone for lymphoma onset. Data show a strong genetic cooperation between the Eµ-myc transgene and SB transposon mobilization with accelerated tumor onset. Arising lymphomas were of the pre/pro- and immature B-cell stage and infiltration included extra-lymphatic tissue like the liver. The genomic sequences immediately adjacent to integrated transposons of 184 lymphomas were enriched by ligation-mediated PCR and were sequenced with a multiplexed approach. Based on published (Brett et al., 2011) and new bioinformatic methods, we identify 338 common integration sites (CIS) of which 188 were found mutated in human B-cell lymphoma. Pathway and GO term analysis reveal modulation of the Ras-MAP-kinase signaling pathway. Next to well-known modulators of Myc-induced lymphomagenesis including Bcl-XL, p53, ARF and Mdm2, we find CIS that were not yet reported in Eµ-myc lymphoma like Map3K5.
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Péneau, Camille. "Mécanismes moléculaires et conséquences oncogéniques des intégrations du Virus de l’Hépatite B dans les tissus hépatiques." Thesis, Université de Paris (2019-....), 2020. https://theses.md.univ-paris-diderot.fr/PENEAU_Camille_va2.pdf.
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Malgré l’existence d’un vaccin efficace et de traitements supprimant la réplication virale, l’infection par le Virus de l’Hépatite B (VHB) reste une des maladies chroniques les plus fréquentes, avec une mortalité associée dans 39% des cas au développement d’un carcinome hépatocellulaire (CHC), le cancer primitif du foie le plus courant et la troisième cause mondiale de décès par cancer. Le VHB est en effet le facteur de risque principal de survenue d’un CHC, chez des patients ayant généralement une cirrhose du foie induite par l’infection. Cependant, le fait que certains CHC liés au VHB surviennent sans inflammation chronique souligne les propriétés oncogéniques directes de ce virus à ADN, qui peut promouvoir la transformation cellulaire des hépatocytes en s’intégrant dans le génome humain. Ce projet a eu pour but de décrire les formes du VHB présentes dans des tissus hépatiques tumoraux et non-tumoraux de 177 patients majoritairement d’origine africaine et européenne, en utilisant des techniques de capture virale et de séquençage de nouvelle génération, et de caractériser les intégrations du virus en fonction des données génétiques et cliniques des patients. Nous avons montré que les tissus non-tumoraux contiennent plus fréquemment de l’ADN viral réplicatif et un nombre total plus élevé d’insertions, principalement localisées dans des régions de chromatine ouverte mais sans conséquence fonctionnelle directe. Dans les tumeurs en revanche, les intégrations du VHB sont souvent clonales, enrichies à proximité de gènes impliqués dans la carcinogenèse hépatique comme TERT (dans un tiers des CHC liés au VHB), CCNE1, ou KMT2B, et peuvent entraîner directement le développement tumoral en activant ces gènes en cis. Les intégrations du VHB dans CCNA2 ou CCNE1 génèrent par exemple un stress réplicatif et une signature de réarrangements structuraux spécifique, favorisant le développement de CHC agressifs en absence de cirrhose. Nous avons décrit par ailleurs un nouveau mécanisme oncogénique associé aux intégrations du VHB qui repose sur des réarrangements du génome humain délimités par des séquences virales intégrées, qui induisent des altérations du nombre de copies de gènes « driver » situés à distance comme TP53 ou MYC. Nous avons donc approfondi la caractérisation des intégrations virales du VHB dans les CHC, mais également celles du virus adéno-associé (AAV) qui peut également s’intégrer dans l’ADN humain et favoriser le développement tumoral par mutagénèse insertionnelle en altérant les mêmes gènes que le VHB (TERT, CCNA2, CCNE1, KMT2B)Despite the existence of an effective vaccine and of treatments that suppress viral replication, Hepatitis B Virus (HBV) infection remains one of the most frequent chronic diseases. 39% of HBV-related deaths are associated with the development of hepatocellular carcinoma (HCC), the most common primary liver cancer and the third leading cause of cancer death worldwide. HBV is indeed the main risk factor of HCC development in patients who generally already have a liver cirrhosis induced by the infection. However, the fact that some HBV-related HCC occur without chronic inflammation underlines the direct oncogenic properties of this DNA virus, which can promote hepatocyte cell transformation through integration into the human genome. This project aimed to describe the HBV genomes in tumor and non-tumor liver tissues from 177 patients, mostly with African and European origin, using viral capture and next-generation sequencing techniques, and characterized viral integrations according to the genetic and clinical data of the patients. We showed that non-tumor tissues contain more frequently replicating HBV DNA and a higher number of insertions, mainly located in open chromatin regions but without direct functional consequences. In tumors, on the other hand, HBV integrations are often clonal and enriched in proximity of genes involved in hepatocarcinogenesis such as TERT (in one-third of HBV-related HCC), CCNE1, or KMT2B, and can directly lead to tumor development by activating these genes in cis. HBV integrations in CCNA2 or CCNE1, for example, generate replicative stress and a specific signature of structural rearrangements, thus promoting the development of aggressive HCC in the absence of cirrhosis. We also described a novel oncogenic mechanism associated with HBV integrations based on rearrangements of the human genome delimited by integrated viral sequences, which induce copy number alterations of distant "driver" genes such as TP53 or MYC. We have therefore further characterized the viral integrations of HBV in HCC, but also those of the adeno-associated virus (AAV) which can also integrate into human DNA and promote tumor development through insertional mutagenesis by altering the same genes as HBV (TERT, CCNA2, CCNE1, KMT2B)
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Callahan, Jennifer Ware. "Analysis of mutations that suppress transport defects in Escherichia coli /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/10276.
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ZANARDI, FEDERICA. "IDENTIFICATION OF NOVEL NON-HODGKIN B-CELL LYMPHOMA DETERMINANTS THROUGH AN IN VIVO, CELL-SPECIFIC TRANSPOSON MUTAGENESIS SCREENING." Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/155504.
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Lymphomas represent the outgrowth of a clonal population of T- or B- lymphocytes. The majority of human lymphomas arise from mature B-cells recruited into germinal centers (GC). B-cell lymphomas fall within two major groups, Hodgkin and Non-Hodgkin (NH) lymphoma. Despite progress has been made toward the elucidation of the molecular basis of lymphomagenesis, few lymphoma determinants have been, to date, causatively linked to the pathogenesis of B-cell lymphomas.
To identify novel genetic determinants of NHL, which include the most common and aggressive forms of B-cell lymphomas, we made use of a cell-type and stage-specific transposon-based, insertional mutagenesis system dependent on the Sleeping Beauty (SB) transposase. As transposon we used T2/Onc2, engineered to cause overexpression of proto-oncogenes or disrupt tumor suppressor genes, respectively.
To restrict transposon mutagenesis to the mature B-cell compartment, we used conditional Rosa26-SBfl-stop mice, in which SB expression, controlled by the Rosa26 promoter, occurs upon Cre-mediated deletion of a loxP-flanked transcription termination sequence. SB expression was induced in mature and GC B-cells using CD21-Cre or Cγ1-Cre transgenes, respectively. The conditional SB system was also used to accelerate lymphomagenesis in mouse NHL models driven by deregulated Bcl6 and Bcl2 expression.
Insertional mutagenesis in GC B-cells was sufficient to promote the occurrence of clonal B-cell tumors at high frequency. B-cell malignancies developing in compound mutants, represented a rather heterogeneous group of diseases originating from GC B-cells, as revealed by the accumulation of somatic mutations within clonal immunoglobulin gene rearrangements. Histological evaluation of tumors revealed close resemblance to human diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the most common forms of NHL. Also, we found that transposon mutagenesis efficiently cooperated with deregulated Bcl6 and Bcl2 to promote GC-derived DLBCL and FL respectively. High throughput sequencing of transposon integration sites from 29 GC-derived B-cell lymphomas identified 240 genes lying within Common Insertion Sites (CISs). Bioinformatic analysis revealed enrichment for genes belonging to the NF-κB signaling network, commonly deregulated in human NHL and in particular in DLBCL. The putative role of CISs as tumor suppressors or proto-oncogenes was assessed based on orientation and gene distribution of T2/Onc2 insertions. Moreover, analysis of gene-copy gain or loss in human lymphomas, occurrence of somatic mutations in human cancers of different type and expression in NHL contributed to select, among the 240 CISs, the strongest candidates to represent novel NHL determinants. Among them, we found genes controlling B-cell differentiation/identity (Ikaros, Aiolos, Bach2 and Swap-70), survival/apoptosis (NF-κB1, NIK, Traf3 Akt2, Rreb1, Zmat3), proliferation (Raf1), chromatin remodeling (Smarcc1) and protein glycosylation (B4galt1, Man2a).
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Thibonnier, Marie. "Etude de la trans-traduction et du métabolisme des ARN chez helicobacter pylori." Paris 7, 2008. http://www.theses.fr/2008PA077075.
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Helicobacter pylori (Hp) est l'agent étiologique des gastrites et des ulcères gastriques pouvant évoluer vers des pathologies plus graves. Son pouvoir pathogène est lié à sa capacité à coloniser de manière persistante un environnement hostile, l'estomac humain. La trans-traduction est un mécanisme bactérien ubiquitaire de contôle-qualité ; SsrA - un petit ARN stable - et SmpB - son cofacteur protéique - libèrent les ribosomes bloqués sur des ARNm défectueux et étiquette la protéine tronquée favorisant ainsi, sa dégradation. L'exploration du rôle de la trans-traduction chez Hp, particulièrement bien adapté à résister à des conditions hostiles a révélé, de manière surprenante, que les gènes smpB et ssrA étaient essentiels ; l'activité essentielle de la trans-traduction est le recyclage des ribosomes qui est assuré uniquement par le codon de reprise de la traduction « résume ». L'étiquetage des protéines est requis pour la résistance à un stress oxydant ou à des concentrations sub-létales en antibiotique. Chez Hp, la stabilité de SsrA augmente en conditions acides similaires à celles rencontrées dans l'estomac. Le contrôle de la stabilité des ARNm joue un rôle clé dans la modulation de l'expression génique, cependant ce type de régulation demeure inexploré chez Hp. Chez Hp, HP1430 est une endoribonucléase essentielle, orthologue à RNaseJ de B. Subtilis. Les partenaires protéiques de HP1430 suggère qu'elle ferait partie d'un dégradosome à l'instar la RNaseE, son homologue fonctionnel chez E. Coll. Cette enzyme fortement régulée - son expression est drastiquement diminuée à pH acide - pourrait être centrale dans le contrôle de la stabilité des ARN et petits ARN chez HpHelicobacter pylori (Hp) is the etiologic agent of the chronic gastritis, gastric ulcers that may evolve in worse pathologies. H. Pylori pathogenesis is linked with its capacity to persistently colonize an hostile niche, the human gastric mucosa. Traws-translation is an ubiquitous quality-control mechanism; SsrA - a stable small RNA - and SmpB - its protein cofactor - freed ribosomes blocked on truncated mRNA and tagged the incomplete protein to direct it to proteolysis pathways. Exploration of the trans-translation in Hp, revealed surprisingly that both smpB and ssrA genes are essential; the essential function is the ribosome recycling. This function relies only on the resume codon. In Hp, peptide tagging is required to resist to oxidative stress or sub-lethal concentration of antibiotics. In Hp, SsrA stability increases in acid conditions similar to those encountered in the stomach. Stability control of thé mRNA plays a key role in the modulation of gene expression, however this field of investigation remain unexplored in Hp. In Hp, HP1430 is an essential endoribonuclease that is orthologous to RNase J of B. Subtilis. Proteins partners of HP1430 suggest that it might be part of a degradosome as RNase E, its functional homolog, does in E. Coll. This enzyme strongly regulated - its expression is drastically diminished at low pH - might play an important role in stability control of mRNA and small RNA in H. Pylori
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Sveric, Krunoslav Michael [Verfasser], and Till [Akademischer Betreuer] Roenneberg. "Novel strategies for the identification of clock genes in Neurospora crassa with insertional mutagenesis / Krunoslav Michael Sveric. Betreuer: Till Roenneberg." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1060005565/34.
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Brett, Benjamin Thomas. "A computational approach for comparative oncogenomics using mouse models." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4582.
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Cancer is the second most common cause of death in the United States. It is a complex disease with environmental, genetic, and lifestyle factors influencing the likelihood of getting cancer and the development of any resulting tumor. Understanding the genetics of cancer is integral to developing novel patient-specific treatments. However, due to complexity, hundreds to thousands of tumors are required for sufficient power to identify the network of relationships among these genes.
Animal models of cancer are commonly used to reduce cost and to control experimental variables allowing for more specific hypothesis testing. The Sleeping Beauty transposon mutagenesis system can be used to model cancer in mice. While the Sleeping Beauty mutagenesis system is an important tool in understanding cancer, it has specific computational needs. Experiments need to be analyzed in a fast, unbiased, and efficient manner. A computational method must also accurately model the system allowing for validation and interpretation. Here I present an updated Integration Analysis System and use this system to validate the assumptions present in forward genetic screens of cancer using the Sleeping Beauty. This system allows for rapid identification of cancer genes, but does not directly aid in understanding the relationship between the genes.
Given the complexity of cancer, understanding the relationship between cancer genes is very difficult. I have created a connectedness network utilizing the STRING database to better derive an understanding of cancer genes. STRING is a database of known and predicted protein-protein interactions. The connectedness between pairs of genes is calculated using a network reliability metric. This database allows for increased power to detect known pathways when compared to STRING alone. Combining this connectivity network with the set of cancer genes identified by the Integration Analysis System is a strategy for rapid and efficient interpretation of the genetic results.
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Castellane, Tereza Cristina Luque [UNESP]. "Obtenção e avaliação de mutantes exoZ- e phbAB- envolvidos no metabolismo de polímeros de carbono em Rhizobium tropici SEMIA 4080 com potencial biotecnológico." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/102842.
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Rhizobium tropici e outras bactérias pertencentes à ordem Rhizobiales são produtores de polissacarídeos extracelulares (EPS), que possuem a função de molécula receptora do micro simbionte, fazendo uma interação célula/célula e desencadeando o processo de nodulação. A diversidade de estruturas e composição química apresentadas pelas moléculas de EPS é refletida pela diversidade de enzimas responsáveis pela sua síntese. Neste trabalho, buscou-se a inativação através da mutagênese insercional por transposição in vitro dos genes exoZ da estirpe selvagem Rhizobium tropici SEMIA 4080 envolvidas na síntese das subunidades repetitivas do EPS e no tanden phbAB, responsáveis pela síntese do PHB, com o intuito de obter microrganismos com aspecto altamente mucoso, produtor superior de EPS em relação a estirpe selvagem. Os mutantes obtidos apresentaram colônias mucosas quando cultivados nos meios de culturas PSYA, demonstrando que estirpes de rizóbio mutantes nos genes exoZ e phbAB são capazes de formar biofilme “in vitro” e, ao avaliar a eficiência relativa na produção de EPS, o mutante 4080 Z03 apresentou o melhor resultado. Para as três amostras de EPS foi possível observar um comportamento de fluxo de líquidos pseudoplástico e, também, a influência da concentração de EPS sobre a viscosidade aparente das soluções aquosas. A estirpe selvagem e todos os mutantes induziram a formação de nódulos em feijoeiro (fenótipo Fix+), sugerindo que os genes exoZ e phbAB não estão envolvidos nos processos de infecção e formação nodular mas, sendo necessários para a fixação biológica de nitrogênio
Rhizobium tropici and other bacteria belonging to the order Rhizobiales are extracellular polysaccharides (EPS) producers, which possess the function of the receptor molecule to function as micro-symbiont, making an interaction cell / cell and triggering the nodulation process. The diversity of structures and chemical composition of EPS presented by molecules is reflected by the diversity of enzymes responsible for its synthesis. In this study, we sought to inactivation by insertional mutagenesis by “in vitro” transposition of genes from wild type exoZ Rhizobium tropici SEMIA 4080 involved in the synthesis of the EPS repeating subunits and phbAB tanden, responsible for synthesis of PHB, in order to obtain microrganisms with high mucosal aspect producer of EPS compared to wild type. The mutants showed mucous colonies when grown in culture media PsyA, demonstrating that mutant strains in genes exoZ and phbAB from rhizobia are able to form biofilm “in vitro”, and to evaluate the relative efficiency in the production of EPS, the mutant 4080 Z03 showed the best result. For the three samples of EPS was possible to observe a flow behavior of pseudoplastic fluids, and also the influence of EPS concentration on the apparent viscosity of aqueous solutions. The wild type and mutants induced the formation of nodules in common bean (Fix+ phenotype), suggesting that genes exoZ and phbAB are not involved in the processes of infection and nodule formation, but are necessary for nitrogen fixation
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Li, Meng. "A recessive genetic screen to discover components in miRNA pathways using piggyBac-mediated insertional mutagenesis in Blm-deficient mouse embryonic stem cells." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609197.
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Plourde, Karine. "L'utilisation de la mutagénèse insertionnelle afin d'identifier des gènes procurant un avantage parasitaire au champignon "Ophiostoma novo-ulmi subsp. novo-ulmi", agent de la maladie hollandaise de l'orme." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27399/27399.pdf.
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Sultana, Tania. "L'influence du contexte génomique sur la sélection du site d'intégration par les rétrotransposons humains L1." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4133.
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Les rétrotransposons L1 (Long INterspersed Element-1) sont des éléments génétiques mobiles dont l'activité contribue à la dynamique du génome humain par mutagenèse insertionnelle. Les conséquences génétiques et épigénétiques d'une nouvelle insertion, et la capacité d'un L1 à être remobilisé, sont directement liées au site d’intégration dans le génome. Aussi, l’analyse des sites d’intégration des L1s est capitale pour comprendre leur impact fonctionnel - voire pathogène -, en particulier lors de la tumorigenèse ou au cours du vieillissement, et l’évolution de notre génome. Dans ce but, nous avons induit de façon expérimentale la rétrotransposition d'un élément L1 actif plasmidique dans des cellules en culture. Puis, nous avons cartographié les insertions obtenues de novo dans le génome humain grâce à une méthode de séquençage à haut-débit, appelée ATLAS-seq. Finalement, les sites pré-intégratifs identifiés par cette approche ont été analysés en relation avec un grand jeu de données publiques regroupant les caractéristiques structurales, génétiques ou épigénétiques de ces loci. Ces expériences ont révélé que les éléments L1 s’intègrent préférentiellement dans des régions de la chromatine faiblement exprimées et renfermant des activateurs faibles. Nous avons aussi trouvé plusieurs positions chromosomiques qui constituent des points chauds d'intégrations récurrentes. Nos résultats indiquent que la distribution des insertions de L1 de novo n’est pas aléatoire, que ce soit à l’échelle chromosomique ou à plus petite échelle, et ouvrent la porte à l'identification des déterminants moléculaires qui contrôlent la distribution chromosomique des L1s dans notre génomeRetrotransposons are mobile genetic elements that employ an RNA intermediate and a reverse transcription step for their replication. Long INterspersed Elements-1 (LINE-1 or L1) form the only autonomously active retrotransposon family in humans. Although most copies are defective due to the accumulation of mutations, each individual genome contains an average of 100 retrotransposition-competent L1 copies, which contribute to the dynamics of contemporary human genomes. L1 integration sites in the host genome directly determine the genetic consequences of the integration and the fate of the integrated copy. Thus, where L1 integrates in the genome, and whether this process is random, is critical to our understanding of human genome evolution, somatic genome plasticity in cancer and aging, and host-parasite interactions. To characterize L1 insertion sites, rather than studying endogenous L1 which have been subjected to evolutionary selective pressure, we induced de novo L1 retrotransposition by transfecting a plasmid-borne active L1 element into HeLa S3 cells. Then, we mapped de novo insertions in the human genome at nucleotide resolution by a dedicated deep-sequencing approach, named ATLAS-seq. Finally, de novo insertions were examined for their proximity towards a large number of genomic features. We found that L1 preferentially integrates in the lowly-expressed and weak enhancer chromatin segments. We also detected several hotspots of recurrent L1 integration. Our results indicate that the distribution of de novo L1 insertions is non-random both at local and regional scales, and pave the way to identify potential cellular factors involved in the targeting of L1 insertions
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Barquist, Lars. "High-throughput experimental and computational studies of bacterial evolution." Thesis, University of Cambridge, 2014. https://www.repository.cam.ac.uk/handle/1810/245138.
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The work in this thesis is concerned with the study of bacterial adaptation on short and long timescales. In the first section, consisting of three chapters, I describe a recently developed high-throughput technology for probing gene function, transposon-insertion sequencing, and its application to the study of functional differences between two important human pathogens, Salmonella enterica subspecies enterica serovars Typhi and Typhimurium. In a first study, I use transposon-insertion sequencing to probe differences in gene requirements during growth on rich laboratory media, revealing differences in serovar requirements for genes involved in iron-utilization and cell-surface structure biogenesis, as well as in requirements for non-coding RNA. In a second study I more directly probe the genomic features responsible for differences in serovar pathogenicity by analyzing transposon-insertion sequencing data produced following a two hour infection of human macrophage, revealing large differences in the selective pressures felt by these two closely related serovars in the same environment. The second section, consisting of two chapters, uses statistical models of sequence variation, i.e. covariance models, to examine the evolution of intrinsic termination across the bacterial kingdom. A first collaborative study provides background and motivation in the form of a method for identifying Rho-independent terminators using covariance models built from deep alignments of experimentally-verified terminators from Escherichia coli and Bacillus subtilis. In the course of the development of this method I discovered a novel putative intrinsic terminator in Mycobacterium tuberculosis. In the final chapter, I extend this approach to de novo discovery of intrinsic termination motifs across the bacterial phylogeny. I present evidence for lineage-specific variations in canonical Rho-independent terminator composition, as well as discover seven non-canonical putative termination motifs. Using a collection of publicly available RNA-seq datasets, I provide evidence for the function of some of these elements as bona fide transcriptional attenuators.
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Guimaraes-Young, Amy. "Gynecological tissue homeostasis and tumorigenesis studies using mouse models." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5942.
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Gynecological cancers present a tremendous disease burden worldwide. Endometrial cancer, the most common gynecological malignancy, is predominantly a disease of deranged glandular function. The mechanisms by which known environmental risk factors influence the mutational profile of endometrial cancer are poorly understood. Non-HPV vulvar cancer, on the other hand, is a very rare gynecological malignancy of vulvar squamous cells with little known about its pathogenesis. Surgical resection of vulvar cancer is associated with high post-surgical morbidity. Pivotal to improving treatment and outcomes for patients with gynecological cancers is an understanding of the molecular drivers unique to each tumor type.
To inform our understanding of endometrial gland regulation, I began my investigations with an assessment of normal endometrial adenogenesis in vivo and present the first evidence implicating the necessity of Sox17 in endometrial gland development. My data suggest Sox17 mediates adenogenesis via a non-cell autonomous mechanism from within the stromal compartment of the endometrium. I then interrogated the contribution of SOX17 to dysregulated glandular function in Type I endometrial adenocarcinoma in vitro. My findings reveal an oncogenic role of SOX17 in the Ishikawa Type 1 endometrial cancer cell line, with homozygous loss of SOX17 impairing cellular proliferation, blunting the cancer phenotype of these cells.
The majority of cancers, including gynecological cancers, develop from the accumulation of genetic mutations that occur sporadically in cells over time. The complexity and heterogeneity of solid tumors, however, renders the identification of mutations responsible for driving tumorigenesis difficult. The Sleeping Beauty (SB) insertional mutagenesis system can be used to streamline sporadic tumor formation and driver mutation identification. I present results from an initial attempt to develop an SB model of endometrial cancer and discuss ways in which the SB system can be harnessed to evaluate tumorigenesis in a variety of tissue types and microenvironmental contexts.
Finally, I present an SB model of metastatic vulvar cancer. Primary tumors from this model resulted in the identification of 76 novel candidate drivers of vulvar cancer, with the ubiquitin-specific peptidase, Usp9x, the most commonly disrupted gene in our screen. I show data suggesting that differential expression of Usp9x isoforms may underlie Usp9x-mediated tumorigenesis and preliminary data demonstrating the relevance of USP9X to human vulvar cancer.
Taken as a whole, these data contribute to our scientific understanding of gynecological tissue homeostasis and cancers, lay the foundation for the development of an SB model of endometrial cancer, and describe the first reported model system for studying HPV-naive vulvar cancer in vivo.
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Foissac, Xavier. "Insertion du Transposon TN4001 dans le génome de Spiroplasma Citri : sélection d'un mutant non transmissible par la Cicadelle Circulifer Haematoceps et d'un mutant non phytopathogene : contribution à l'étude de la spiraline protéine majeure de la membrane des spiroplasmes du groupe I." Bordeaux 2, 1995. http://www.theses.fr/1995BOR28367.
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Chami, Mounia. "Impact de la mutagénèse insertionnelle liée au virus de l'hépatite B dans la carcinogenèse hépatique : implication des protéines SERCA1 tronquées dans le processus d'apoptose." Paris 11, 2001. http://www.theses.fr/2001PA11T006.
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L'impact de la mutagenèse insertionnelle dans les mécanismes de carcinogenèse hépatique liée au virus de l'hépatite B (VHB) a été considéré pendant longtemps comme anécdotique. Nous avons appliqué la technique PCR-Alu afin d'analyser les sites d'intégration du génome viral dans le génome cellulaire et de définir l'impact réel de la mutagenèse insertionelle liée au VHB dans les mécanismes de carcinogenèse hépatique. Cette étude nous a permis d'isoler 21 jonctions ADN du VHB/ADN cellulaire à partir de 18 tumeurs. Dans cinq jonctions issues de cinq tumeurs différentes, l'ADN du VHB est intégré : 1) dans un nouveau gène de la famille TRAP (TRAP150 Thyroid Hormone Receptor Associated Protein 150), 2) dans un nouveau gène de la famille MCM (2/3/5) (Minichromosome Maintenance protein), 3) dans un nouveau gène non encore caractérisé, 4) dans le gène SERCA1 (Sarco Endoplasmic Reticulum Calcium ATP ase) et 5) en amont du promoteur du gène codant pour la telomerase hTERT (Human Telomerase Reverse Transcriptase). Nos résultats valorisent l'impact de la mutagenèse insertionnelle au cours de l'hépatocarcinogenèse et révèlent de nouveaux gènes potentiellement impliqués dans la tumorigenèse. Dans le cadre de l'analyse des sites d'intégration, le génome du VHB peut servir de sonde pour rechercher de nouveaux gènes et mécanismes impliqués dans la carcinogenèse hépatique. La suite de mon travail a été axée sur la caractérisation de l'intégration du VHB dans le gène SERCA1. Nous avons pu montrer la surexpression des ARN et des protéines hybrides dans la zone tumorale par rapport à la zone non tumorale. Les transcrits hybrides X VHB/SERCA1 sont constitués par une séquence X du VHB en 5' et une séquence SERCA1 en 3' caractérisée par un épissage alternatif de l'exon 4 et/ou de l'exon 11 donnant naissance à un codon stop prématuré au niveau de l'exon 12. Notre travail a pu également démontrer l'expression de ces deux nouveaux transcrits SERCA1 non chimériques dans différents tissus humains adultes et fœtaux. Ces transcrits codent pour des protéines SERCA1 tronquées en C terminale (S1T) incapables de fonctionner comme des pompes calciques. Les protéines hybrides et S1T montrent une localisation au niveau du RE et s'accumulent en homodimères dans la fraction microsomale des cellules. Leur expression in vitro induit une diminution de la concentration du calcium dans le RE associée à une augmentation de la fuite passive du calcium du RE vers le cytoplasme. L'expression des protéines hybrides et S1T induit également une apoptose dans trois lignées cellulaires d'origine hépatique. Cette deuxième partie de mon travail a permis de révéler un nouveau mécanisme de régulation de l'homéostasie calcique du RE et de suggérer son rôle dans le processus d'apoptose et de transformation cellulaire.
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Castellane, Tereza Cristina Luque. "Obtenção e avaliação de mutantes exoZ- e phbAB- envolvidos no metabolismo de polímeros de carbono em Rhizobium tropici SEMIA 4080 com potencial biotecnológico /." Jaboticabal : [s.n.], 2011. http://hdl.handle.net/11449/102842.
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Resumo: Rhizobium tropici e outras bactérias pertencentes à ordem Rhizobiales são produtores de polissacarídeos extracelulares (EPS), que possuem a função de molécula receptora do micro simbionte, fazendo uma interação célula/célula e desencadeando o processo de nodulação. A diversidade de estruturas e composição química apresentadas pelas moléculas de EPS é refletida pela diversidade de enzimas responsáveis pela sua síntese. Neste trabalho, buscou-se a inativação através da mutagênese insercional por transposição in vitro dos genes exoZ da estirpe selvagem Rhizobium tropici SEMIA 4080 envolvidas na síntese das subunidades repetitivas do EPS e no tanden phbAB, responsáveis pela síntese do PHB, com o intuito de obter microrganismos com aspecto altamente mucoso, produtor superior de EPS em relação a estirpe selvagem. Os mutantes obtidos apresentaram colônias mucosas quando cultivados nos meios de culturas PSYA, demonstrando que estirpes de rizóbio mutantes nos genes exoZ e phbAB são capazes de formar biofilme "in vitro" e, ao avaliar a eficiência relativa na produção de EPS, o mutante 4080 Z03 apresentou o melhor resultado. Para as três amostras de EPS foi possível observar um comportamento de fluxo de líquidos pseudoplástico e, também, a influência da concentração de EPS sobre a viscosidade aparente das soluções aquosas. A estirpe selvagem e todos os mutantes induziram a formação de nódulos em feijoeiro (fenótipo Fix+), sugerindo que os genes exoZ e phbAB não estão envolvidos nos processos de infecção e formação nodular mas, sendo necessários para a fixação biológica de nitrogênioAbstract: Rhizobium tropici and other bacteria belonging to the order Rhizobiales are extracellular polysaccharides (EPS) producers, which possess the function of the receptor molecule to function as micro-symbiont, making an interaction cell / cell and triggering the nodulation process. The diversity of structures and chemical composition of EPS presented by molecules is reflected by the diversity of enzymes responsible for its synthesis. In this study, we sought to inactivation by insertional mutagenesis by "in vitro" transposition of genes from wild type exoZ Rhizobium tropici SEMIA 4080 involved in the synthesis of the EPS repeating subunits and phbAB tanden, responsible for synthesis of PHB, in order to obtain microrganisms with high mucosal aspect producer of EPS compared to wild type. The mutants showed mucous colonies when grown in culture media PsyA, demonstrating that mutant strains in genes exoZ and phbAB from rhizobia are able to form biofilm "in vitro", and to evaluate the relative efficiency in the production of EPS, the mutant 4080 Z03 showed the best result. For the three samples of EPS was possible to observe a flow behavior of pseudoplastic fluids, and also the influence of EPS concentration on the apparent viscosity of aqueous solutions. The wild type and mutants induced the formation of nodules in common bean (Fix+ phenotype), suggesting that genes exoZ and phbAB are not involved in the processes of infection and nodule formation, but are necessary for nitrogen fixation
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Coorientador: Manoel Victor Franco Lemos
Banca: Regiane de Fátima Travensolo Sacomano
Banca: Simone Cristina Picchi
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Duplay, Pascale. "Approche genetique de la topologie fonctionnelle de la proteine affine du maltose chez escherichia coli." Paris 7, 1987. http://www.theses.fr/1987PA077001.
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Nelson, Bryn D. "Examining the role of MalG in the assembly and function of the maltose transport complex in Escherichia coli : implications for the study of integral membrane proteins /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/11508.
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La, Bella Tiziana. "Adeno-associated virus in the liver : natural history of the infection and consequences in tumor development." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC263.
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Le virus adéno-associé (AAV) est un virus à ADN monobrin, défectif et endémique dans la population humaine. L'infection à AAV a longtemps été considérée comme non pathogénique. Cependant, il y a quelques années, nous avons identifié pour la première fois des insertions clonales récurrentes d'AAV2 impliquées dans la pathogenèse du carcinome hépatocellulaire humain (CHC) développé sur un foie normal. Ces insertions virales clonales ciblent des oncogènes et entraînent leur surexpression. À ce jour, peu de travaux ont étudié l'infection à AAV de type sauvage dans le foie humain. Dans ce travail, nous avons étudié l'histoire naturelle de l'infection virale dans les tissus hépatiques et ses conséquences sur le développement de tumeurs dans une vaste cohorte de patients (n = 1464). La présence de l'AAV est observée chez 21% des patients, plus fréquemment dans la contrepartie non-tumorale (18%) que dans la tumeur (8%) et significativement enrichie chez les femmes, les patients jeunes et non cirrhotiques. Deux sous-types d’AAV ont été identifiés dans le foie, l’AAV2 classique et un génotype hybride AAV2-AAV3-AAV13, avec une fréquence égale dans notre cohorte. Nous avons détecté la présence de formes épisomales d'AAV dans 27% des tissus non tumoraux positifs pour l'AAV, associée de manière significative à l'expression de l'ARN viral et à la co-infection par des virus auxiliaires, suggérant une infection active en cours. Nous avons identifié le virus de l'herpès humain de type 6 (HHV6) comme étant le virus auxiliaire naturel de l'AAV dans le foie. En revanche, l'ADN de l'adénovirus n'a été détecté que chez 0,5% des patients et aucune association avec l'AAV n'a été constatée. Nous avons confirmé la sélection positive des insertions d'AAV clonales au cours du développement du CHC chez les patients sans cirrhose dans 2% des tumeurs ciblant CCNA2, CCNE1, TERT, TNFSF10, KMT2B et INHBE / GLI1. De plus, les altérations de CCNA2 et de CCNE1 dues à des insertions virales d'AAV et de VHB ou à des réarrangements structurels ont défini une nouvelle sous-classe de CHC (CCN-HCC) et un nouveau mécanisme de développement du CHC sur le foie normal, améliorant nos connaissances sur l'hépatocarcinogénèse sur le foie non cirrhotique. Les CCN-HCC présentent également des caractéristiques moléculaires particulières qui pourraient être ciblées par un traitement spécifiqueAdeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population. AAV infection has long been considered as non-pathogenic, however few years ago we reported for the first time recurrent clonal AAV2 insertion in the pathogenesis of human hepatocellular carcinoma (HCC) developed on normal liver. These clonal viral insertions target cancer driver genes leading to their overexpression. To date, little is known about wild type AAV infection in human liver. In this work we investigated the natural history of the viral infection in the liver tissues and the consequences in tumor development in a large cohort of patients (n=1464). The presence of AAV was observed in 21% of patients, more frequently in the non-tumor counterpart (18%) than in tumor (8%) and significantly enriched in young, female and non-cirrhotic patients. Two AAV subtypes were identified in the liver, the classical AAV2 and a hybrid AAV2-AAV3-AAV13 genotypes, with an equal frequency in our cohort. We detected the presence of episomal AAV forms in 27% of AAV positive non-tumor tissues significantly associated with viral RNA expression and co-infection with helper viruses suggesting an ongoing active infection. We identified human herpes virus type 6 (HHV6) as the natural AAV helper virus in the liver. In contrast, adenovirus DNA was detected in only 0.5% of patients and no association with AAV was found. We confirmed the positive selection of clonal AAV insertions during HCC development in patients without cirrhosis in 2% of tumors targeting CCNA2, CCNE1, TERT, TNFSF10, KMT2B and INHBE/GLI1. Moreover, the alterations in CCNA2 and CCNE1 due to viral insertions of AAV and HBV or structural rearrangements defined a new subclass of HCCs (CCN-HCC) and a novel mechanism of HCC development on normal liver improving our knowledge on hepatocarcinogenesis on non-cirrhotic liver. CCN-HCCs display also peculiar molecular features that could be targeted by specific treatment
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NOIRCLERC, MARJOLAINE. "Transposition de sequences d'insertion (is) chez la bacterie escherichia coli." Université Joseph Fourier (Grenoble), 2001. http://www.theses.fr/2001GRE10041.
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La comparaison de la nature de 1674 mutants obtenus dans plusieurs contextes genetiques et conditions physiologiques, a montre que la mobilite des sequences is n'etait pas constante au cours de la croissance bacterienne. En milieu riche, la transposition des is est totalement absente en phase de croissance exponentielle, et ne se produit qu'en phase stationnaire. Ce phenomene semble etre independant du facteur de transcription rpos, specifique de la phase stationnaire. Cependant, la transposition peut se produire en phase de croissance exponentielle dans un milieu ou la croissance est ralentie par des carences nutritionnelles. Nous avons aussi identifie une region chromosomique impliquee dans la transposition. La deletion de cette region permet la mobilisation d'is1 et d'is5 des la phase de croissance exponentielle, mais il n'a pas ete possible d'affecter ce phenomene a un gene unique de cette region. L'hypothese formulee a l'issue de ce travail est que la transposition de certaines is est favorisee, lorsque le bon fonctionnement cellulaire est altere. Des donnees recentes montrent par exemple que le dysfonctionnement du ribosome en fonction des ressources nutritives augmente la transposition d'is1.